Use of mtor inhibitors for prevention of intestinal polyp growth and cancer

ABSTRACT

Disclosed are methods and compositions for the treatment or prevention of intestinal polyps or prevention of cancer in a patient who has been identified as being at risk for developing intestinal polyps or intestinal cancer. The disclosed methods and compositions include rapamycin, a rapamycin analog, or another such inhibitor of the target of rapamycin (TOR).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalApplication No. 61/778,670, filed on Mar. 13, 2013, which is herebyincorporated by reference in its entirety.

GOVERNMENTAL RIGHTS

This invention was made with government support under agreement numberAG036613 awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

A. Field of the Invention

The invention relates to methods and compositions for treating orpreventing intestinal adenomas or polyps or preventing cancer in apatient who has been identified as being at risk for developingintestinal polyps or intestinal cancer. The methods and compositionsinclude rapamycin, rapamycin analogs, or other inhibitors of themammalian target of rapamycin (“mTOR” or “mTORC1”).

B. Description of Related Art

Intestinal cancer encompasses a variety of cancers, including cancer ofthe small intestine, gastric cancer, and colorectal cancer.Historically, the most common treatment of intestinal cancer is surgeryand radiation therapy. To date, there are no effective therapies forprevention of intestinal cancers. Rather, most treatments focus on earlydetection and treatment of existing cancers.

There are three main roles that chemoprevention can play in colorectalcancer (CRC) patients: (1) to delay prophylactic colectomy; (2) toprevent cancer development in the retained rectum in patients aftercolectomy with ileorectal anastomosis (IRA); and (3) to prevent cancerdevelopment in the upper gastrointestinal tract, especially theduodenum. Nonsteroidal anti-inflammatory drugs (NSAIDs) includingsulindac and celecoxib, ursodeoxycholic acid, statins,difluoromethylornithine (DFMO), and various dietary supplements havebeen studied as potential chemopreventive agents (Kim 2011). Thenon-selective, non-steroidal anti-inflammatory drug (NSAID) sulindac canbe given to delay the progression of polyposis in the retained rectumamong patients after colectomy with IRA but should be used inconjunction with a strict endoscopic surveillance regimen (Giardiello1993; Labayle 1991; Rigau 1991). In a 6-month trial in adults with FAP,the selective cyclooxygenase inhibitor celecoxib was found to be welltolerated and significantly reduced colorectal adenomas (Steinbach2000). Sulindac or celecoxib is not recommended as a primarychemopreventive agent. Despite apparent effectiveness, reports ofpotential cardiovascular toxicity with COX-2 inhibitors limit their usein FAP (Solomon 2005).

The benefit of regular use of COX-2 inhibitors and non-selective NSAIDsin FAP patients with cardiovascular risk factors needs to be weighedagainst the potential cardiovascular adverse events of thesemedications. Cardiovascular complications do not appear to be limited toCOX-2 inhibitor use. Non-selective NSAIDs, including sulindac andnaproxen, have been suggested to increase cardiovascular thromboticevents (Zell 2009). Chemoprevention should ideally be well tolerated,low in toxicity, inexpensive, and effective for long-term use.

In view of this, there remains a need for therapies that preventintestinal cancer and treat underlying symptoms that lead to cancer.

SUMMARY OF THE INVENTION

In some aspects, provided are methods for preventing intestinal polypsor intestinal cancer in a patient comprising administering an effectiveamount of a composition comprising rapamycin or an analog thereof to apatient who has been identified as being at risk for developingintestinal polyps or intestinal cancer.

In some embodiments, the patient has been identified as being at riskfor developing intestinal polyps or intestinal cancer. In someembodiments, this risk is identified on the basis of disease state,prior diagnosis, family history, diet, age, or other factors. In someembodiments, the patient has been diagnosed with an inflammatory boweldisease. In some embodiments, the patient has been diagnosed with anintestinal polyp or an adenoma. In some embodiments, the patient hasbeen diagnosed as having a mutation that is known to cause increased WNTsignaling. In some embodiments, the patient has been diagnosed as havingFamilial Adenomatous Polyposis (FAP). In some embodiments, the patienthas a family history of intestinal polyps or intestinal cancer. In someembodiments, the patient is between the ages of 1 to 18 years, 18 yearsto 50 years, or over the age of 50 years.

In some embodiments, the rapamycin or analog thereof is encapsulated orcoated, or the composition comprising the rapamycin or analog thereof isencapsulated or coated. In some embodiments, the encapsulant or coatingmay be an enteric coating. In some embodiments, the encapsulant orcoating may be an enteric coating. In some embodiments, the coatingcomprises cellulose acetate succinate, hydroxy propyl methyl cellulosephthalate co-polymer, or a polymethacrylate-based copolymer selectedfrom the group consisting of methyl acrylate-methacrylic acid copolymer,and a methyl methacrylate-methacrylic acid copolymer. In someembodiments, the coating comprises Poly(methacylic acid-co-ethylacrylate) in a 1:1 ratio, Poly(methacrylic acid-co-ethyl acrylate) in a1:1 ratio, Poly(methacylic acid-co-methyl methacrylate) in a 1:1 ratio,Poly(methacylic acid-co-methyl methacrylate) in a 1:2 ratio, Poly(methylacrylate-co-methyl methacrylate-co-methacrylic acid) in a 7:3:1 ratio,Poly(ethyl acrylate-co-methyl methacrylate-co-trimethylammonioethylmethacrylate chloride) in a 1:2:0.2 ratio, Poly(ethyl acrylate-co-methylmethacrylate-co-trimethylammonioethyl methacrylate chloride) in a1:2:0.1 ratio, or Poly(butyl methacylate-co-(2-dimethylaminoethyl)methacrylate-co-methyl methacrylate) in a 1:2:1 ratio, anaturally-derived polymer, or a synthetic polymer, or any combinationthereof. In some embodiments, the naturally-derived polymer is selectedfrom the group consisting of alginates and their various derivatives,chitosans and their various derivatives, carrageenans and their variousanalogues, celluloses, gums, gelatins, pectins, and gellans. In someembodiments, the naturally-derived polymer is selected from the groupconsisting of polyethyleneglycols (PEGs) and polyethyleneoxides (PEOs),acrylic acid homo- and copolymers with acrylates and methacrylates,homopolymers of acrylates and methacrylates, polyvinyl alcohol PVOH),and polyvinyl pyrrolidone (PVP).

An effective amount of rapamycin or rapamycin analog or derivative willdepend upon the disease to be treated, the length of duration desiredand the bioavailability profile of the implant, and the site ofadministration. In some embodiments, the composition comprises rapamycinor an analog thereof at a concentration of 0.001 mg to 30 mg total perdose. In some embodiments, the composition comprising rapamycin or ananalog of rapamycin comprises 0.001% to 60% by weight of rapamycin or ananalog of rapamycin. In some embodiments, the average blood level ofrapamycin in the subject is greater than 0.5 ng per mL whole blood afteradministration of the composition.

The composition can be administered to the subject using any methodknown to those of ordinary skill in the art. In some embodiments, thecomposition may be administered intravenously, intracerebrally,intracranially, intraventricularly, intrathecally, into the cortex,thalamus, hypothalamus, hippocampus, basal ganglia, substantia nigra orthe region of the substantia nigra, cerebellum, intradermally,intraarterially, intraperitoneally, intralesionally, intratracheally,intranasally, topically, intramuscularly, intraperitoneally, anally,subcutaneously, orally, topically, locally, inhalation (e.g., aerosolinhalation), injection, infusion, continuous infusion, localizedperfusion bathing target cells directly, via a catheter, via a lavage,in creams, in lipid compositions (e.g., liposomes), or by other methodsor any combination of the forgoing as would be known to one of ordinaryskill in the art. In some embodiments, the composition is administeredorally, enterically, colonically, anally, intravenously, or dermallywith a patch. In some embodiments, the composition comprising rapamycinor an analog of rapamycin is comprised in a food or food additive.

The dose can be repeated as needed as determined by those of ordinaryskill in the art. In some embodiments, the rapamycin or analog ofrapamycin is administered in two or more doses. Where more than one doseis administered to a subject, the time interval between doses can be anytime interval as determined by those of ordinary skill in the art. Forexample, the two doses may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 29, 20, 21, 22, 23, or 24 hours apart, or any rangetherein. In some embodiments, the composition may be administered daily,weekly, monthly, annually, or any range therein. In some embodiments,the interval of time between administration of doses comprisingrapamycin or an analog of rapamycin is between 0.5 to 30 days.

In some embodiments, the method comprises further administering one ormore secondary or additional forms of therapies. In some embodiments,the subject is further administered a composition comprising a secondactive agent. In some embodiments, the second active agent is metformin,celocoxib, eflornithine, sulindac, ursodeoxycholic acid, ananti-inflammatory agent, an anti-autoimmune agent, or a cytotoxic orcytostatic anti-cancer agent. In some embodiments, the compositioncomprising rapamycin or an analog of rapamycin is administered at thesame time as the composition comprising the second active agent. In someembodiments, the composition comprising rapamycin or an analog ofrapamycin is administered before or after the composition comprising thesecond active agent is administered. In some embodiments, the twotreatments may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 29, 20, 21, 22, 23, or 24 hours apart, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 29, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, or 31 days apart, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or12 months apart, or one or more years apart or any range therein. Insome embodiments, the interval of time between administration ofcomposition comprising rapamycin or an analog of rapamycin and thecomposition comprising the second active agent is 1 to 30 days.

In some embodiments, the composition comprising rapamycin or an analogof rapamycin prevents intestinal polyps or intestinal cancer. In someembodiments, the composition comprising rapamycin or an analog ofrapamycin prevents the development of new adenomas or polyps, decreasesthe number or severity of the adenomatous polyps, induces a reduction insize or number of existing adenomas or polyps, prevents the conversionof adenomas or polyps into adenocarcinomas and cancer tissue, orprevents the adenomas or polyps from converting into malignant cancerthat spread into other bodily tissues, organs and blood systems in apatient that has been diagnosed as having intestinal adenomas,intestinal polyps or Familial Adenomatous Polyposis (FAP).

In some embodiments, the mTOR inhibitor or an analog thereof is eRapa.“eRapa” is generically used to refer to encapsulated or coated forms ofRapamycin or other mTOR inhibitors or their respective analogs disclosedherein and equivalents thereof. In some embodiments, the encapsulant orcoating used for and incorporated in eRapa preparation may be an entericcoating. In some embodiments, the mTOR inhibitor or analog thereof isnanoRapa. “nanoRapa” is generically used to refer to the rapamycins,rapamycin analogs, or related compositions within the eRapa preparationare provided in the form of nanoparticles that include the rapamycin orother mTOR inhibitor. In some embodiments, the mTOR inhibitor or analogthereof is e-nanoRapa. “e-nanoRapa” is generically used to refer toeRapa variations formed from nanoRapa particles. After preparing thenanoRapa preparations, the nanoRapa preparation may then be coated withan enteric coating, to provide an eRapa preparation formed from nanoRapaparticles.

In some embodiments, the eRapa, nanoRapa, or e-nanoRapa is encased in acoating comprising cellulose acetate succinate, hydroxy propyl methylcellulose phthalate co-polymer, or a polymethacrylate-based copolymerselected from the group consisting of methyl acrylate-methacrylic acidcopolymer, and a methyl methacrylate-methacrylic acid copolymer. In someembodiments, the coating comprises Poly(methacylic acid-co-ethylacrylate) in a 1:1 ratio, Poly(methacrylic acid-co-ethyl acrylate) in a1:1 ratio, Poly(methacylic acid-co-methyl methacrylate) in a 1:1 ratio,Poly(methacylic acid-co-methyl methacrylate) in a 1:2 ratio, Poly(methylacrylate-co-methyl methacrylate-co-methacrylic acid) in a 7:3:1 ratio,Poly(ethyl acrylate-co-methyl methacrylate-co-trimethylammonioethylmethacrylate chloride) in a 1:2:0.2 ratio, Poly(ethyl acrylate-co-methylmethacrylate-co-trimethylammonioethyl methacrylate chloride) in a1:2:0.1 ratio, or Poly(butyl methacylate-co-(2-dimethylaminoethyl)methacrylate-co-methyl methacrylate) in a 1:2:1 ratio, anaturally-derived polymer, or a synthetic polymer, or any combinationthereof. In some embodiments, the naturally-derived polymer is selectedfrom the group consisting of alginates and their various derivatives,chitosans and their various derivatives, carrageenans and their variousanalogues, celluloses, gums, gelatins, pectins, and gellans. In someembodiments, the naturally-derived polymer is selected from the groupconsisting of polyethyleneglycols (PEGs) and polyethyleneoxides (PEOs),acrylic acid homo- and copolymers with acrylates and methacrylates,homopolymers of acrylates and methacrylates, polyvinyl alcohol PVOH),and polyvinyl pyrrolidone (PVP).

In some embodiments, the composition comprises eRapa or an analogthereof at a concentration of at or between 50 micrograms and 200micrograms per kilogram for daily administration, or the equivalent forother frequencies of administration.

In some embodiments, the eRapa, nanoRapa, or e-nanoRapa is administeredorally, enterically, colonically, anally, intravenously, or dermallywith a patch. In some embodiments, the eRapa, nanoRapa, or e-nanoRapa isadministered in two or more doses. In some embodiments, the interval oftime between administration of doses comprising eRapa, nanoRapa, ore-nanoRapa is 0.5 to 30 days. In some embodiments, the interval of timebetween administration of doses comprising eRapa, nanoRapa, ore-nanoRapa is 0.5 to 1 day. In some embodiments, the interval of timebetween administration of doses comprising eRapa, nanoRapa, ore-nanoRapa is 1 to 3 days. In some embodiments, the interval of timebetween administration of doses comprising eRapa, nanoRapa, ore-nanoRapa is 1 to 5 days. In some embodiments, the interval of timebetween administration of doses comprising eRapa, nanoRapa, ore-nanoRapa is 1 to 7 days. In some embodiments, the interval of timebetween administration of doses comprising eRapa, nanoRapa, ore-nanoRapa is 1 to 15 days.

In some embodiments, the subject is further administered a compositioncomprising a second active agent. In some embodiments, the second activeagent is metformin, celocoxib, eflornithine, sulindac, ursodeoxycholicacid, an anti-inflammatory agent, an anti-autoimmune agent, or acytotoxic or cytostatic anti-cancer agent. In some embodiments, thecomposition comprising eRapa, nanoRapa, or e-nanoRapa is administered atthe same time as the composition comprising the second active agent. Insome embodiments, the composition comprising eRapa, nanoRapa, ore-nanoRapa is administered before or after the composition comprisingthe second active agent is administered. In some embodiments, theinterval of time between administration of composition comprising eRapa,nanoRapa, or e-nanoRapa and the composition comprising the second activeagent is 1 to 30 days.

In some embodiments, the composition comprising eRapa, nanoRapa, ore-nanoRapa prevents intestinal polyps or intestinal cancer. In someembodiments, the composition comprising eRapa, nanoRapa, or e-nanoRapaprevents the development of new adenomas or polyps, decreases the numberor severity of the adenomatous polyps, induces a reduction in size ornumber of existing adenomas or polyps, prevents the conversion ofadenomas or polyps into adenocarcinomas and cancer tissue, or preventsthe adenomas or polyps from converting into malignant cancer that spreadinto other bodily tissues, organs and blood systems in a patient thathas been diagnosed as having intestinal adenomas, intestinal polyps orFamilial Adenomatous Polyposis (FAP).

In some embodiments, the composition comprising eRapa, nanoRapa, ore-nanoRapa is comprised in a food or food additive.

Unless otherwise specified, the percent values expressed herein areweight by weight and are in relation to the total composition.

The term “about” or “approximately” is defined as being close to asunderstood by one of ordinary skill in the art, and in one non-limitingembodiment the terms are defined to be within 10%, preferably within 5%,more preferably within 1%, and most preferably within 0.5%.

The terms “inhibiting,” “reducing,” “treating,” or any variation ofthese terms, includes any measurable decrease or complete inhibition toachieve a desired result. Similarly, the term “effective” means adequateto accomplish a desired, expected, or intended result.

The terms “prevention” or “preventing” includes: (1) inhibiting theonset of a disease in a subject or patient which may be at risk and/orpredisposed to the disease but does not yet experience or display any orall of the pathology or symptomatology of the disease, and/or (2)slowing the onset of the pathology or symptomatology of a disease in asubject or patient which may be at risk and/or predisposed to thedisease but does not yet experience or display any or all of thepathology or symptomatology of the disease.

The use of the word “a” or “an” when used in conjunction with the term“comprising” may mean “one,” but it is also consistent with the meaningof “one or more,” “at least one,” and “one or more than one.”

The words “comprising” (and any form of comprising, such as “comprise”and “comprises”), “having” (and any form of having, such as “have” and“has”), “including” (and any form of including, such as “includes” and“include”) or “containing” (and any form of containing, such as“contains” and “contain”) are inclusive or open-ended and do not excludeadditional, unrecited elements or method steps. in relation to the totalcomposition.

The compositions and methods for their use can “comprise,” “consistessentially of,” or “consist of” any of the ingredients or stepsdisclosed throughout the specification. With respect to the transitionalphrase “consisting essentially of,” in one non-limiting aspect, a basicand novel characteristic of the compositions and methods is the abilityof eRapa, e-nanoRapa or other rapamycin preparations to treat or preventintestinal polyps or prevent cancer in a patient who has been identifiedas being at risk for developing intestinal polyps or intestinal cancer,most especially in subjects who are suspected or known to have a geneticpredisposition for developing familial adenomatous polyposis (FAP).

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method or composition of theinvention, and vice versa. Furthermore, compositions of the inventioncan be used to achieve methods of the invention.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-1B Encapsulated rapamycin increases life span and health inApc^(Min/+) mice. (A) Encapsulated Rapa increases life span inApc^(Min/+) mice. (B) Life span of rapamycin-treated Apc^(Min/+) micecompared to wild type C57B6 mice or mice treated with RAD001(everolimus).

FIGS. 2A-2C (A) Polyp count in Apc^(Min/+) mice at time of death in micetreated with no eRapa (left cluster, solid circles), the medium dose ofeRapa as described above (middle cluster, solid squares), or the highdose of eRapa as described above (right cluster, solid triangles). (B)Encapsulated Rapa improves physical activity in Apc^(Min/+) mice, asshown by measuring the average number of beam breaks (activity) for twotime periods of the day, light and dark. Mice were treated with Eudragitcontrol diet (left column, hatched), the medium dose of eRapa asdescribed above (middle column, white with black dots), or the high doseof eRapa as described above (right column, black with white dots) Thefood area of the cage was excluded. (C) Encapsulated Rapa maintainsnormal hematocrits in Apc^(Min/+) mice. Mice were treated with no eRapa(solid black circles), the medium dose of eRapa (solid black squares),or the high dose of eRapa (solid black triangles). Age at the time ofhematocrits is indicated on the X-axis.

FIGS. 3A-3C. Encapsulated Rapamycin inhibits mTOR complex 1 (mTORC1)downstream effector, ribosomal protein subunit S6 (rpS6) phosphorylationby S6 kinase 1 (S6K1) in the distal segment of small intestine. eRapawas fed to C57BL/6 mice, the same genetic background for theApc^(Min/+), and intestines were collected and prepared for immunoassay.(A) Immunoblot showing detection of total rpS6 (bottom panel),Ser240/244 phosphorylated rpS6 (middle panel) and pan actin as a loadingcontrol. (B) Signal intensities for each band in (A) were quantified andthe ratio of phosphorylated rpS6 to total rpS6 was calculated andgraphed as a scatter plot using Prism Software. Statistical significanceof the reduction in this ratio was determined using an un-paired t-test(Prism). These data show that eRapa effectively inhibited, mTORC1 andits effector rpS6, which is known to play a vital role in biogenesis ofribosomes used in protein synthesis needed for cell growth andproliferation. This is likely the effect of eRapa that inhibits polypdevelopment and growth in Apc^(Min/+) mice and extends longevity. (C)Blood levels of rapamycin at 217 days (174 days treatment with eRapa) inmice treated with 14 ppm eRapa (the medium dose of eRapa as describedabove, see bottom cluster, shown in solid black squares) and 42 ppmeRapa (the high dose of eRapa as described above, see top cluster, shownin solid black circles).

FIG. 4 depicts an embodiment of methods of the present invention,showing a sequence of steps for producing nanoRapa rapamycinnanoparticles by stirring a mixture of a combination of rapamycin and awater-miscible solvent with a combination of water and dispersants.

FIG. 5 depicts an embodiment of methods of the present invention,showing a sequence of steps for producing e-nanoRapa microencapsulatednanoparticles of rapamycin.

FIG. 6 depicts a nanoRapa embodiment illustrating a detailed view of amicelle created by particular dispersants in solution as is used as partof a sequence of fabricating the nanoRapa rapamycin nanoparticles ofFIG. 1.

FIG. 7 depicts particular e-nanoRapa embodiments of the invention,particularly with reference to fabrication of e-nanoRapamicroencapsulated nanoparticles of rapamycin as produced by the methodof FIG. 5.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The inventors have discovered an effective therapy for treating orpreventing intestinal polyps or preventing cancer in a patient who hasbeen identified as being at risk for developing intestinal polyps orintestinal cancer comprising administration of rapamycin, an analog ofrapamycin, or another inhibitor of mTOR. In certain embodiments, therapamycin, an analog of rapamycin, or other inhibitor of mTOR isadministered orally. In certain embodiments, the rapamycin, an analog ofrapamycin, or other inhibitor of mTOR is administered in the form of aneRapa and/or e-nanoRapa preparation.

Chemoprevention should ideally be well tolerated, low in toxicity,inexpensive, and effective for long-term use; therefore, thecompositions disclosed herein appear to be ideal for this purpose. Asshown in FIG. 1-2, encapsulated rapamycin demonstrates significantpotential in preventing, delaying and/or reducing the severity ofintestinal polyps, as evidenced by prevention of anemia (FIG. 2C, 3C).

A. Intestinal Cancer

Intestinal cancer encompasses a variety of cancers, including cancer ofthe small intestine, gastric cancer, and colorectal cancer. Symptoms ofintestinal cancer often include, but are not limited to, pain throughoutthe body, unexplained weight-loss, pain or cramping in the middle of theabdomen, a lump in the abdomen, blood in the stool, nausea, bloating,iron deficient anemia, and jaundice. Historically, the most commontreatment of intestinal cancer is surgery and radiation therapy.

Intestinal cancer is more likely to occur in some patients than others.For example, intestinal cancer is more likely to occur in a patient thathas been diagnosed with an inflammatory bowel disease, an intestinalpolyp or an adenoma, Familial adenomatous polyposis (FAP), or as havinga mutation which is known to cause increased WNT signaling. In otherembodiments, the patient has a family history of intestinal polyps orintestinal cancer.

Small intestine cancer can be further divided into a variety ofsubtypes, including cancer of the jejunum and ileum, duodenal cancer,adenocarcinoma, gastrointestinal stromal tumors, lymphoma, and ilealcarinoid tumors. Adenocarcinoma is a type of cancer that begins in thelining of the small intestine, and make up 40-50% of all smallintestinal cancers. This type of intestinal cancer occurs most oftenlater in life. People with Crohn's Disease and certain other inheritedconditions such as familial adenomatous polyposis andPeuts-Jegherssyndrome are at a higher risk of developing adenocarinomas.Carcinoid tumors occur when neuroendocrine cells grow abnormally, andmay also be referred to as neuroendocrine tumors or neuroendocrinecancer. People with a family history of multiple endocrine neoplasia ora family history of neurofibromatosis are more likely to get carcinoidtumors. Carcinoid tumors are also more common in women, AfricanAmericans, and people with certain diseases that damage the stomach andreduce the amount of stomach acid. Gastrointestinal stromal tumors startin the interstitial cells of Cajal (ICCs) in the walls of the GI tract.It is believed that a family history of neurofibromatosis or familialgastrointestinal stromal tumor syndrome will increase a patient's riskof getting stromal tumors. Gastrointestinal lymphomas are a cancer ofthe lymphatic system that begins in the lymphoid tissue. It is believedthat old age, genetic risk factors that cause abnormal function of theimmune system, a diet high in animal fat and low in fruits andvegetables, exposure to radiation and certain chemicals, immunedeficiencies, and some infections increase the likelihood of a lymphomadeveloping.

Colorectal cancer, commonly also known as colon cancer or bowel cancer,is a cancer from uncontrolled cell growth in the colon, rectum, orappendix. The majority of colorectal cancers are due to lifestyle andincreasing age, but some are associated with an underlying geneticdisorder. For example, people with inflammatory bowel disease(ulcerative colitis and Crohn's disease) are at increased risk of coloncancer. Those with a family history of colorectal cancer in two or morefirst-degree relatives have a two to threefold greater risk of disease,and a number of genetic syndromes are also associated with higher ratesof colorectal cancer. The most common of these is hereditarynonpolyposis colorectal cancer (HNPCC or Lynch syndrome) which ispresent in about 3% of people with colorectal cancer. Other syndromesthat are strongly associated include: Gardner syndrome, and familialadenomatous polyposis (FAP).

Gastric cancer refers to cancer arising from any part of the stomach,and is often either asymptomatic or causes only nonspecific symptoms inits early stages. Infection by Helicobacter pylori is believed to be thecause of most stomach cancer while autoimmune atrophic gastritis,intestinal metaplasia, and various genetic factors are associated withincreased risk levels. A very important but preventable cause of gastriccancer is tobacco smoking Gastric cancers due to smoking mostly occur inthe upper part of the stomach near the esophagus.

B. Familial Adenomatous Polyposis (FAP)

Familial adenomatous polyposis (FAP) is an autosomal dominant diseasecaused by mutation of the Adenomatous Polyposis Coli (APC) gene, locatedon chromosome 5 (Kinzler 1991). This germline defect accelerates theinitiation of the adenoma-carcinoma, resulting in the development ofnumerous adenomatous colorectal polyps at a young age. Polyposisinevitably progresses to colorectal cancer if left untreated. Given thepredictable development of colorectal cancer in patients with FAP, thesafest preventative strategy is surgical resection of the colon whenpolyposis develops. The two main prophylactic surgeries are colectomywith ileorectal anastamosis (IRA) and proctocolectomy with ilealpouch-anal anastamosis (IPAA) (Vasen 2008). Genetic screening andendoscopy in concert with prophylactic surgery significantly improvedthe overall survival of FAP patients. A pharmacological prophylacticapproach to prevent these outcomes for this population of patients isobviously in great need.

However, less well appreciated is the second leading cause of death inFAP, duodenal adenocarcinoma. Nearly 90% of patients with FAP developduodenal polyps, the precursor lesions of duodenal adenocarcinoma and4.5% will develop duodenal adenocarcinoma in their lifetime (Wallace1998; Bulow 2004). In contrast to the colon, prophylactic surgicalresection of the ampulla and/or duodenum is accompanied by significantmorbidity. Duodenal surgery is currently indicated for patients withsevere duodenal polyposis or duodenal carcinoma. This patient populationhas a strong need for adjuvant therapies to surgery to prevent or reducethe polyp formation and carcinogenesis in the gastro-intestinal track.

C. WNT Signaling Pathway

WNTs comprise a family of 19 secreted glycoproteins, which function indiverse biological processes such as cell proliferation, survival andsegment polarity during development (Anastas 2013). WNTs signal viatransmembrane receptors included in 10 members of the frizzled (FZD)family of G-protein coupled receptors and receptor tyrosine kinases. Thefirst WNT gene was identified in cancer arising in mouse models ofmammary cancer and in mouse and human colon cancer. WNTs promotestabilization of a transcription factor called β-catenin (also known asCTNNB1). WNTs control both the canonical β-catenin-dependent andnon-canonical (β-catenin-independent pathways. Studies point to a vitalrole for hyper-activated WNT-β-catenin signaling in colorectal cancer(Korinek 1997; Morin 1997) Inherited inactivating mutations of theadenomatous polyposis coli (APC) gene, the product of which is anegative controller of β-catenin stability, are found in patients withfamilial adenomatous polyposis (FAP). Polyps of FAP patients progress tocolorectal carcinomas upon inactivation of the tumor suppressor p53 andactivating mutations of KRAS. Both APC and CTNNB1 are commonly mutatedin colorectal cancers of non-FAP patients.

The high prevalence of WNT pathway mutations in many types of cancer isevidence for the importance of the WNT-β-catenin pathway incarcinogenesis. Mutations in other members of the WNT signal pathwayimplicated in carcinogenesis include: TCF7L2 (transcription factor7-like), CTNNB1, WTX (Wilms tumor gene on the X chromosome), and AXIN(See Table 1 of Anastas 2013)

D. mTOR Inhibitors and Rapamycin

Any inhibitor of mTORC1 is contemplated for inclusion in the presentcompositions and methods. In particular embodiments, the inhibitor ofmTORC1 is rapamycin or an analog of rapamycin. In some embodiments, theinhibitor of mTORC1 is rapamycin or an analog of rapamycin isadministered orally in the form of an eRapa and/or e-nanoRapapreparation. Rapamycin (also known as sirolimus and marketed under thetrade name Rapamune) is a known macrolide. The molecular formula ofrapamycin is C₅₁H₇₉NO₁₃.

Rapamycin binds to a member of the FK binding protein (FKBP) family,FKBP 12. The rapamycin/FKBP 12 complex binds to the protein kinase mTORto block the activity of signal transduction pathways. Because the mTORsignaling network includes multiple tumor suppressor genes, includingPTEN, LKB1, TSC1, and TSC2, and multiple proto-oncogenes including PI3K,Akt, and eEF4E, mTOR signaling plays a central role in cell survival andproliferation. Binding of the rapamycin/FKBP complex to mTOR causesarrest of the cell cycle in the G1 phase (Janus 2005).

mTORC1 inhibitors also include rapamycin analogs. Many rapamycin analogsare known in the art. Non-limiting examples of analogs of rapamycininclude, but are not limited to, everolimus, tacrolimus, CCI-779,ABT-578, AP-23675, AP-23573, AP-23841, 7-epi-rapamycin,7-thiomethyl-rapamycin, 7-epi-trimethoxyphenyl-rapamycin,7-epi-thiomethyl-rapamycin, 7-demethoxy-rapamycin,32-demethoxy-rapamycin, 2-desmethyl-rapamycin, and 42-O-(2-hydroxy)ethylrapamycin.

Other analogs of rapamycin include: rapamycin oximes (U.S. Pat. No.5,446,048); rapamycin aminoesters (U.S. Pat. No. 5,130,307); rapamycindialdehydes (U.S. Pat. No. 6,680,330); rapamycin 29-enols (U.S. Pat. No.6,677,357); O-alkylated rapamycin derivatives (U.S. Pat. No. 6,440,990);water soluble rapamycin esters (U.S. Pat. No. 5,955,457); alkylatedrapamycin derivatives (U.S. Pat. No. 5,922,730); rapamycin amidinocarbamates (U.S. Pat. No. 5,637,590); biotin esters of rapamycin (U.S.Pat. No. 5,504,091); carbamates of rapamycin (U.S. Pat. No. 5,567,709);rapamycin hydroxyesters (U.S. Pat. No. 5,362,718); rapamycin42-sulfonates and 42-(N-carbalkoxy)sulfamates (U.S. Pat. No. 5,346,893);rapamycin oxepane isomers (U.S. Pat. No. 5,344,833); imidazolidylrapamycin derivatives (U.S. Pat. No. 5,310,903); rapamycin alkoxyesters(U.S. Pat. No. 5,233,036); rapamycin pyrazoles (U.S. Pat. No.5,164,399); acyl derivatives of rapamycin (U.S. Pat. No. 4,316,885);reduction products of rapamycin (U.S. Pat. Nos. 5,102,876 and5,138,051); rapamycin amide esters (U.S. Pat. No. 5,118,677); rapamycinfluorinated esters (U.S. Pat. No. 5,100,883); rapamycin acetals (U.S.Pat. No. 5,151,413); oxorapamycins (U.S. Pat. No. 6,399,625); andrapamycin silyl ethers (U.S. Pat. No. 5,120,842).

Other analogs of rapamycin include those described in U.S. Pat. Nos.6,015,809; 6,004,973; 5,985,890; 5,955,457; 5,922,730; 5,912,253;5,780,462; 5,665,772; 5,637,590; 5,567,709; 5,563,145; 5,559,122;5,559,120; 5,559,119; 5,559,112; 5,550,133; 5,541,192; 5,541,191;5,532,355; 5,530,121; 5,530,007; 5,525,610; 5,521,194; 5,519,031;5,516,780; 5,508,399; 5,508,290; 5,508,286; 5,508,285; 5,504,291;5,504,204; 5,491,231; 5,489,680; 5,489,595; 5,488,054; 5,486,524;5,486,523; 5,486,522; 5,484,791; 5,484,790; 5,480,989; 5,480,988;5,463,048; 5,446,048; 5,434,260; 5,411,967; 5,391,730; 5,389,639;5,385,910; 5,385,909; 5,385,908; 5,378,836; 5,378,696; 5,373,014;5,362,718; 5,358,944; 5,346,893; 5,344,833; 5,302,584; 5,262,424;5,262,423; 5,260,300; 5,260,299; 5,233,036; 5,221,740; 5,221,670;5,202,332; 5,194,447; 5,177,203; 5,169,851; 5,164,399; 5,162,333;5,151,413; 5,138,051; 5,130,307; 5,120,842; 5,120,727; 5,120,726;5,120,725; 5,118,678; 5,118,677; 5,100,883; 5,023,264; 5,023,263;5,023,262; all of which are incorporated herein by reference. Additionalrapamycin analogs and derivatives can be found in the following U.S.Patent Application Pub. Nos., all of which are herein specificallyincorporated by reference: 20080249123, 20080188511; 20080182867;20080091008; 20080085880; 20080069797; 20070280992; 20070225313;20070203172; 20070203171; 20070203170; 20070203169; 20070203168;20070142423; 20060264453; and 20040010002.

Rapamycin or a rapamycin analog can be obtained from any source known tothose of ordinary skill in the art. The source may be a commercialsource, or natural source. Rapamycin or a rapamycin analog may bechemically synthesized using any technique known to those of ordinaryskill in the art. Non-limiting examples of information concerningrapamycin synthesis can be found in Schwecke et al., 1995; Gregory etal., 2004; Gregory et al., 2006; Graziani, 2009.

E. Encapsulated Rapamycin Compositions

In some aspects, the compositions comprising an inhibitor of mTOR areencapsulated or coated to provide eRapa preparations. In someembodiments, the encapsulant or coating may be an enteric coating. Insome embodiments, the compositions comprising an inhibitor of mTOR areprovided in the form of nanoRapa nanoparticles, and such nanoRapananoparticles are encapsulated or coated to provide e-nanoRapapreparations, which are relatively stable and beneficial for oraladministration.

Many pharmaceutical dosage forms irritate the stomach due to theirchemical properties or are degraded by stomach acid through the actionof enzymes, thus becoming less effective. The coating may be an entericcoating, a coating that prevents release and absorption of activeingredients until they reach the intestine. “Enteric” refers to thesmall intestine, and therefore enteric coatings facilitate delivery ofagents to the small intestine. Some enteric coatings facilitate deliveryof agents to the colon. In some embodiments, the enteric coating is aEUDRAGIT(®) coating. Eudragit coatings include Eudragit L100-55 (fordelivery to the duodenum), Poly(methacylic acid-co-ethyl acrylate) 1:1;Eudragit L 30 D-55 (for delivery to the duodenum), Poly(methacrylicacid-co-ethyl acrylate) 1:1; Eudragit L 100 (for delivery to thejejunum), Poly(methacylic acid-co-methyl methacrylate) 1:1; EudragitS100 (for delivery to the ileum), Poly(methacylic acid-co-methylmethacrylate) 1:2; Eudragit FS 30D (for colon delivery), Poly(methylacrylate-co-methyl methacrylate-co-methacrylic acid) 7:3:1; Eudragit RL(for sustained release), Poly(ethyl acrylate-co-methylmethacrylate-co-trimethylammonioethyl methacrylate chloride) 1:2:0.2;Eudragit RS (for sustained release), Poly(ethyl acrylate-co-methylmethacrylate-co-trimethylammonioethyl methacrylate chloride) 1:2:0.1;and Eudragit E (for taste masking), Poly(butylmethacylate-co-(2-dimethylamino ethyl) methacrylate-co-methylmethacrylate) 1:2:1. Other coatings include Eudragit RS, Eudragit RL,ethylcellulose, and polyvinyl acetate. Benefits include pH-dependentdrug release, protection of active agents sensitive to gastric fluid,protection of gastric mucosa from active agents, increase in drugeffectiveness, good storage stability, and GI and colon targeting, whichminimizes risks associated with negative systemic effects.

Some examples of enteric coating components include cellulose acetatepthalate, methyl acrylate-methacrylic acid copolymers, cellulose acetatesuccinate, hydroxy propyl methyl cellulose phthalate, hydroxy propylmethyl cellulose acetate succinate, polyvinyl acetate phthalate, methylmethacrylate-methacrylic acid copolymers, sodium alginate, and stearicacid. The coating may include suitable hydrophilic gelling polymersincluding but not limited to cellulosic polymers, such asmethylcellulose, carboxymethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, hydroxyethylcellulose, and the like; vinylpolymers, such as polyvinylpyrrolidone, polyvinyl alcohol, and the like;acrylic polymers and copolymers, such as acrylic acid polymer,methacrylic acid copolymers, ethyl acrylate-methyl methacrylatecopolymers, natural and synthetic gums, such as guar gum, arabic gum,xanthan gum, gelatin, collagen, proteins, polysaccharides, such aspectin, pectic acid, alginic acid, sodium alginate, polyaminoacids,polyalcohols, polyglycols; and the like; and mixtures thereof. Any othercoating agent known to those of ordinary skill in the art iscontemplated for inclusion in the coatings of the microcapsules setforth herein.

The coating may optionally comprises a plastisizer, such as dibutylsebacate, polyethylene glycol and polypropylene glycol, dibutylphthalate, diethyl phthalate, triethyl citrate, tributyl citrate,acetylated monoglyceride, acetyl tributyl citrate, triacetin, dimethylphthalate, benzyl benzoate, butyl and/or glycol esters of fatty acids,refined mineral oils, oleic acid, castor oil, corn oil, camphor,glycerol and sorbitol or a combination thereof. The coating mayoptionally include a gum. Non-limiting examples of gums includehomopolysaccharides such as locust bean gum, galactans, mannans,vegetable gums such as alginates, gum karaya, pectin, agar, tragacanth,accacia, carrageenan, tragacanth, chitosan, agar, alginic acid, otherpolysaccharide gums (e.g., hydrocolloids), acacia catechu, salai guggal,indian bodellum, copaiba gum, asafetida, cambi gum, Enterolobiumcyclocarpum, mastic gum, benzoin gum, sandarac, gambier gum, buteafrondosa (Flame of Forest Gum), myrrh, konjak mannan, guar gum, welangum, gellan gum, tara gum, locust bean gum, carageenan gum, glucomannan,galactan gum, sodium alginate, tragacanth, chitosan, xanthan gum,deacetylated xanthan gum, pectin, sodium polypectate, gluten, karayagum, tamarind gum, ghatti gum, Accaroid/Yacca/Red gum, dammar gum,juniper gum, ester gum, ipil-ipil seed gum, gum talha (acacia seyal),and cultured plant cell gums including those of the plants of thegenera: acacia, actinidia, aptenia, carbobrotus, chickorium, cucumis,glycine, hibiscus, hordeum, letuca, lycopersicon, malus, medicago,mesembryanthemum, oryza, panicum, phalaris, phleum, poliathus,polycarbophil, sida, solanum, trifolium, trigonella, Afzelia africanaseed gum, Treculia africana gum, detarium gum, cassia gum, carob gum,Prosopis africana gum, Colocassia esulenta gum, Hakea gibbosa gum, khayagum, scleroglucan, zea, mixtures of any of the foregoing, and the like.

In some aspects, the compositions comprising an inhibitor of mTOR areformed into nanoparticles and subsequently encapsulated or coated. Insome embodiments, the encapsulant or coating may be an enteric coating.In some embodiments, the encapsulated rapamycin nanoparticles providerapamycin nanoparticles within a protective polymer matrix for oraladministration of rapamycin. The result is not only more durable andstable, but is also more bioavailable and efficacious for treatment andprevention of genetically-predisposed disorders and age-relateddisorders, especially in the fields of oncology and neurology in humansand other animals.

The encapsulated rapamycin nanoparticles provide an embodiment of thepresent invention in the form of an improved form of encapsulatedrapamycin that is more durable, stable and bioavailable. In someembodiments, the encapsulated rapamycin provides the rapamycinnanoparticles within a controlled release matrix, forming theencapsulated rapamycin nanoparticle in a single drug delivery structurefor oral administration of rapamycin. This encapsulated rapamycinnanoparticle may also be referred to as an enteric-coated rapamycinnanoparticle. In addition, many of the embodiments also include astabilizing compound (for our purposes, a “stabilizer”) within thecontrolled release matrix either to improve compatibility of therapamycin with the controlled release matrix, to stabilize thecrystalline morphology of the rapamycin, or to help further preventdegradation of the rapamycin, particularly when the encapsulatedrapamycin nanoparticle is exposed to air, atmospheric moisture, or roomtemperature or warmer conditions. Particular embodiments incorporate thestabilizers within each rapamycin nanoparticle, although certain aspectsof the invention may be embodied with stabilizers on the surface of theencapsulated rapamycin nanoparticles or otherwise dispersed in thecontrolled release matrix. To different levels depending on theparticular approach used for producing the nanoparticles, with orwithout other additives, the result is more efficacious for treatmentand prevention of genetically-predisposed disorders and age-relateddisorders, especially in the fields of oncology and neurology in humansand other animals.

Rapid anti-solvent precipitation, or controlled precipitation, is onemethod of preparing the rapamycin nanoparticles as it provides forminimal manipulation of the rapamycin and exquisite control overnanoparticle size and distribution, and the crystallinity of therapamycin. Several controlled precipitation methods are known in theart, including rapid solvent exchange and rapid expansion ofsupercritical solutions, both of which can be implemented in batch orcontinuous modes, are scalable, and suitable for handling pharmaceuticalcompounds.

Rapamycin nanoparticles prepared by controlled precipitation methods canbe stabilized against irreversible aggregation, Ostwald ripening, and/orreduced dispersibility, by control of colloid chemistry, particlesurface chemistry and particle morphology. For example, nanoparticlesprepared by antisolvent solidification can be stabilized by ionic andnon-ionic surfactants that adsorb to nanoparticle surfaces and promoteparticle colloid stability through either charge repulsion or sterichindrance, respectively. Moreover, stabilizers can affect nanoparticlecrystallinity, which may be used to promote different biodistributionand bioavailability in certain indications.

Rapamycin nanoparticles can consist of molecular rapamycin bound bysuitable methods to other nanoparticles. Suitable methods of attachingrapamycin to a nanoparticle carrier or substrate may include physicaladsorption through hydrogen van der Waals forces or chemisorptionthrough covalent or ionic bonding. Nanoparticle substrates may be eithernatural or synthetic, and modified to promote specific interactions withrapamycin. Natural nanoparticles include albumin and other proteins, andDNA. Synthetic nanoparticles include organic and inorganic particulates,micelles, liposomes, dendrimers, hyperbranched polymers, and othercompounds.

The rapamycin nanoparticles can be processed by any suitable method,such as by milling, high-pressure atomization, or rapid anti-solventprecipitation. Milling is suitable provided care is taken to minimizeboth rapamycin degradation and particle agglomeration. Rapamycindegradation can be reduced with the aid of cooling or cryogenicprocesses. Agglomeration due to the increased surface area andconcomitant adhesive forces can be reduced by the use of dispersantsduring the milling process.

In some embodiments, the rapamycin nanoparticles are sized between about1 nanometer and about 1 micron. In some embodiments, the rapamycinnanoparticles are less than 1 micron diameter. Such smaller particlesprovide better control of final particle size, improved stability withinthe particles, and the ability to tune bioavailability by controllingthe crystallinity and composition of the rapamycin nanoparticles.

Manufacturing approaches for the encapsulated rapamycin nanoparticledrug delivery structure embodiments of the present invention includecreating a solution of the controlled release matrix, with the rapamycinnanoparticles dispersed therein, in appropriate proportion and producinga heterogeneous mixture. The solvent for such mixtures can be a suitablevolatile solvent for the controlled release matrix. In some embodiments,the solvent is either a poor solvent or non-solvent for the rapamycinnanoparticles so that when the rapamycin nanoparticles are dispersedinto the controlled release matrix solution they remain as discretenanoparticles. The resulting dispersion of rapamycin nanoparticles inthe controlled release matrix solution can then be reduced to a dryparticulate powder by a suitable process, thereby resulting inmicroparticles of a heterogeneous nature comprised of rapamycinnanoparticles randomly distributed in the controlled release matrix. Theparticulate powder may also be tailored by a suitable process to achievea desired particle size for subsequent preparation, which may be fromabout 20 to about 70 microns in diameter.

The rapamycin nanoparticles are microencapsulated with the controlledrelease matrix using a suitable particle-forming process to form theencapsulated rapamycin nanoparticle. An example of a particle-formingprocess is spinning disk atomization and drying. For a detaileddiscussion of the apparatus and method concerning the aforementionedspin disk coating-process, this application incorporates by referencesUS Patent Applications 2011/221337 and 2011/220430, respectively.Alternatively, for example, the encapsulated rapamycin nanoparticles canbe prepared by spray drying.

In some embodiments, not all of the rapamycin nanoparticles will beencapsulated within the controlled release matrix. Instead the rapamycinnanoparticles may be enmeshed with the controlled release matrix, withsome of the rapamycin nanoparticles wholly contained within thecontrolled release matrix while another other rapamycin nanoparticlesapparent on the surface of the drug delivery structure, constructed inappearance similar to a chocolate chip cookie.

In some embodiments, and depending on the size of the rapamycinnanoparticles, the encapsulated rapamycin nanoparticles are between 10and 50 microns in diameter, although diameters as large as 75 micronsmay be suitable.

The controlled release matrix of the encapsulated rapamycinnanoparticles can be selected to provide desired release characteristicsof the encapsulated rapamycin nanoparticles. For example, the matrix maybe pH sensitive to provide either gastric release or enteric release ofthe rapamycin. Enteric release of the rapamycin may achieve improvedabsorption and bioavailability of the rapamycin. Many materials suitablefor enteric release are known in the art, including fatty acids, waxes,natural and synthetic polymers, shellac, and other materials. Polymersare a one enteric coating and may include copolymers of methacrylic acidand methyl methacrylate, copolymers of methyl acrylate and methacrylicacid, sodium alginate, polyvinyl acetate phthalate, and varioussuccinate or phthalate derivatives of cellulose and hydroxpropyl methylcellulose. Synthetic polymers, such as copolymers of methacrylic acidand either methyl acrylate or methyl methacrlate, are good entericrelease polymers due the ability to tune the dissolution pH range ofthese synthetic polymers by adjusting their comonomer compositions.Examples of such pH sensitive polymers are EUDRAGIT® polymers (EvonikIndustries, Essen, Germany). Specifically, EUDRAGIT® S-100, a methylmethacrylate and methacrylic acid copolymer with comonomer ratio of 2:1,respectively, has a dissolution pH of about 7.0, thereby making issuitable for enteric release of rapamycin.

The encapsulated rapamycin nanoparticles may be delivered in variousphysical entities including a pill, tablet, or capsule. The encapsulatedrapamycin nanoparticles may be pressed or formed into a pellet-likeshape and further encapsulated with a coating, for instance, an entericcoating. In another embodiment, the encapsulated rapamycin nanoparticlesmay be loaded into a capsule, also further enterically coated.

Various performance enhancing additives can be added to the encapsulatedrapamycin nanoparticles. For example, additives that function as freeradical scavengers or stabilizers can be added to improve oxidative andstorage stability of the encapsulated rapamycin nanoparticles. In someembodiments, free radical scavengers are chosen from the group thatconsists of glycerol, propylene glycol, and other lower alcohols.Additives alternatively incorporate antioxidants, such as α-tocopherol(vitamin E), citric acid, EDTA, α-lipoic acid, or the like.

Methacrylic acid copolymers with methyl acrylate or methyl methacrylateare moderate oxygen barriers. Furthermore, these polymers will exhibitan equilibrium moisture content. Oxygen transport due to residualsolvent, moisture or other causes, can lead to degradation of theencapsulated rapamycin nanoparticles. Oxygen barrier materials can beadded to the encapsulated rapamycin nanoparticles formulation to improveoxygen barrier properties. Oxygen barrier polymers compatible with thepolymers are polyvinyl alcohol (PVA) and gelatin.

F. Microparticle and Nanoparticle Rapamycin

In some embodiments, rapamycin nanoparticle inclusions comprise discretenanoparticles of rapamycin heterogeneously dispersed in a controlledrelease matrix. As illustrated in FIGS. 4-6, the rapamycin nanoparticlesare prepared by a suitable method and may contain additives to promotenanoparticle stability, modify rapamycin crystallinity, or promotecompatibility of the rapamycin nanoparticles with the controlled releasematrix. The controlled release matrix is formulated to promote releaseof rapamycin to specific parts of the body, such as the intestine, toenhance oxidative and storage stability of the encapsulated rapamycinnanoparticles, and to maintain the discrete, heterogeneously distributednature of the rapamycin nanoparticles.

Referring to FIG. 4, rapamycin nanoparticles are prepared byanti-solvent precipitation or solidification, also sometimes referred toas controlled precipitation or solidification. Antisolventsolidification is one approach as it provides exquisite control ofparticle size and distribution, particle morphology, and rapamycincrystallinity. For example, it is possible to prepare nanoparticles withnarrow particle size distribution that are amorphous, crystalline, orcombinations thereof. Such properties may exhibit additional benefits,by further controlling the biodistribution and bioavailability ofrapamycin in specific indications.

Referring now to FIG. 5, rapamycin is dissolved in a suitablewater-miscible solvent and then rapidly injected into rapidly stirredwater containing an appropriate aqueous soluble dispersant.Water-miscible solvents for rapamycin include methanol, ethanol,isopropyl alcohol, acetone, dimethylsulfoxide, dimethylacetamide,n-methylpyrolidone, tetrahydrofuran, and other solvents. Low boilingpoint, high vapor pressure water-miscible solvents facilitate theirremoval during subsequent microparticle formation. Examplarywater-miscible solvents are methanol, acetone, and isopropyl alcohol. Insome embodiments, the water-miscible solvent is methanol. Some aqueoussoluble dispersants include ionic surfactants such as sodium dodecylsulfate and sodium cholate, non-ionic surfactants such as Pluronics,Poloxomers, Tweens, and polymers, such as polyvinyl alcohol andpolyvinylpyrolidone. Examplary aqueous-soluble dispersants are sodiumcholate, Pluronic F-68, and Pluronic F-127. In some embodiments, theaqueous-soluble dispersant is sodium cholate, which providessurprisingly beneficial properties. Not only is sodium cholate asurfactant and a dispersant, it serves to cause aggregation of rapamycinparticles from the aqueous solution. Moreover, while sodium cholatetends to be a polar molecule as well as an amphoteric surfactant, itsurrounds each nanoparticle with a hydrophobic charge when it isenmeshed in the Eudragit matrix. Then, when the nanoparticle is releasedfrom the Eudragit matrix within the animal subject's enteric passageswhere conditions are basic, the same properties cause the nanoparticleto be more readily received and absorbed through the intestinal walls.

Referring to FIG. 6 now, rapamycin is dissolved in the water-misciblesolvent at a concentration of about 0.01% w/v to about 10.0% w/vpreferably about 0.1% w/v to about 1.0% w/v. The aqueous-solubledispersant is dissolved in water at a concentration above its criticalmicelle concentration, or CMC, typically at about 1 to about 10 timesthe CMC. The rapamycin solution is injected into the aqueous-solubledispersant solution with agitation at a volumetric ratio of about 1:10to about 1:1, preferably about 1:5 to about 1:1.

The controlled release matrix is prepared from a water-soluble polymer,which may be a copolymer of methacrylic acid with either methyl acrylateor methyl methacrylate, such as those marketed under the trade name ofEUDRAGIT® and having pH-dependent dissolution properties. The controlledrelease matrix may be comprised of EUDRAGIT® S-100, although otherwater-soluble enteric controlled release would be suitable.Water-soluble controlled release matrices are selected so as either notto compromise the integrity of rapamcyin nanoparticles or to provide amedium in which rapamycin nanoparticles may be prepared by thecontrolled precipitation methodology described previously.

In preparing the water-soluble polymer it is helpful to maintainconditions that do not compromise the integrity of the rapamycinnanoparticles. Firstly, since the rapamycin nanoparticles aresusceptible solubilization by certain co-solvents, it is important tomaintain a suitable quantity of certain co-solvents to achievecontrolled release matrix solubility while not deleteriously affectingthe morphology of the rapamycin nanoparticles. Secondly, rapamycinnanoparticles will be susceptible to chemical degradation by high pH;therefore, it is important to modulate the controlled release matrixsolution pH so that rapamycin is not chemically altered. It is helpfulthe controlled release matrix solution pH be maintained below about pH8. Lastly, it is helpful to achieve near to complete solubilization ofthe controlled release matrix in solution so that microencapsulation ofthe rapamycin nanoparticles by the controlled release matrix insubsequent processing steps may proceed with high efficiency. When usingthe EUDRAGIT® S-100 as the controlled release matrix, it is helpful toachieve a controlled release matrix solution by using a combination ofco-solvents and solution pH modulation. In certain embodiments, theco-solvents are about 40% or less by volume. Similarly, in certainembodiments, the pH of the controlled release matrix solution is about 8or less, such that the EUDRAGIT® S-100 is not completely neutralized andmay be only about 80% or less neutralized. These conditions achievenearly complete to complete solubilization of the EUDRAGIT® S-100 in amedium that is mostly aqueous and that maintains the integrity of therapamycin nanoparticles, therefore leading to their microencapsulationby the controlled-release matrix in subsequent processing steps.

The rapamycin nanoparticles prepared by the controlled precipitationmethod are added to the aqueous solution of the controlled releasedmatrix, resulting in a nanoparticle dispersion in the solubilizedcontrolled release matrix. Alternatively, the rapamycin solubilized in asuitable co-solvent can be dispersed into the aqueous solution ofcontrolled release matrix leading to controlled precipitation ofrapamycin particles, thereby leading to a rapamycin nanoparticledispersion in fewer processing steps, but of appropriate composition topermit subsequent microencapsulation processing.

As an alternative embodiment, the encapsulated rapamycin nanoparticlesare created using pre-existing nanoparticle substrates, such as albumin,to create, in the case of albumin, “albumin-rapamycin nanoparticles.”Within this general class of alternatives, certain approaches forcreating the albumin-rapamycin nanoparticles involve encapsulatingrapamycin within albumin nanoparticles or preferentially associatingrapamycin with albumin nanoparticles through physical or chemicaladsorption. The albumin nanoparticles themselves may be formed fromhuman serum albumin, a plasma protein derived from human serum.

More particularly, this embodiment may involve use of a therapeuticpeptide or protein that is covalently or physically bound to albumin, toenhance its stability and half-life. With the albumin stabilized, therapamycin is mixed with the stabilized albumin in an aqueous solvent andpassed under high pressure to form rapamycin-albumin nanoparticles inthe size range of 100-200 nm (comparable to the size of smallliposomes).

Certain embodiments also address degradation risks and other limitsimposed by the related art by preparing encapsulated rapamycinnanoparticles as a heterogeneous mixture of rapamycin nanoparticles in apolymer matrix. Distributed nanoparticles are morphologically differentthan homogeneous rapamycin; and are less susceptible to degradationbecause of the bulk nature of the nanoparticles compared to the smallersize of molecular rapamycin.

G. Methods of Using Rapamycin Compositions

“Treatment” and “treating” refer to administration or application of atherapeutic agent to a subject or performance of a procedure or modalityon a subject for the purpose of obtaining a therapeutic benefit for adisease or health-related condition. For example, the rapamycincompositions of the present invention may be administered to a subjectfor the purpose of treating or preventing intestinal adenomas or polypsand cancer in a patient who has been identified as being at risk fordeveloping intestinal polyps or intestinal cancer.

The terms “therapeutic benefit,” “therapeutically effective,” or“effective amount” refer to the promotion or enhancement of thewell-being of a subject. This includes, but is not limited to, areduction in the frequency or severity of the signs or symptoms of adisease.

“Prevention” and “preventing” are used according to their ordinary andplain meaning. In the context of a particular disease or health-relatedcondition, those terms refer to administration or application of anagent, drug, or remedy to a subject or performance of a procedure ormodality on a subject for the purpose of preventing or delaying theonset of a disease or health-related condition. For example, oneembodiment includes administering the rapamycin compositions of thepresent invention to a subject at risk of developing intestinal polypsand cancer (e.g., a patient who has been diagnosed with FAP) for thepurpose of preventing intestinal polyps and cancer.

Rapamycin compositions, as disclosed herein, may be used to treat anydisease or condition for which an inhibitor of mTOR is contemplated aseffective for treating or preventing the disease or condition. Forexample, methods of using rapamycin compositions to treat or preventintestinal polyps and cancer in a patient who has been identified asbeing at risk for developing intestinal polyps or intestinal cancer aredisclosed. This risk for developing intestinal polyps or intestinalcancer may be determined by genetic analysis. The treatment orprevention of the disease may be instituted before or after any relatedsurgical intervention such as polypectomy or any form of a full orpartial colectomy or colon resection. Dosing regimens may includemultiple doses per day, one dose per day, or regular doses one or moredays apart.

Other uses of rapamycin compositions as disclosed herein are alsocontemplated. For example, U.S. Pat. No. 5,100,899 discloses inhibitionof transplant rejection by rapamycin; U.S. Pat. No. 3,993,749 disclosesrapamycin antifungal properties; U.S. Pat. No. 4,885,171 disclosesantitumor activity of rapamycin against lymphatic leukemia, colon andmammary cancers, melanocarcinoma and ependymoblastoma; U.S. Pat. No.5,206,018 discloses rapamycin treatment of malignant mammary and skincarcinomas, and central nervous system neoplasms; U.S. Pat. No.4,401,653 discloses the use of rapamycin in combination with otheragents in the treatment of tumors; U.S. Pat. No. 5,078,999 discloses amethod of treating systemic lupus erythematosus with rapamycin; U.S.Pat. No. 5,080,899 discloses a method of treating pulmonary inflammationwith rapamycin that is useful in the symptomatic relief of diseases inwhich pulmonary inflammation is a component, i.e., asthma, chronicobstructive pulmonary disease, emphysema, bronchitis, and acuterespiratory distress syndrome; U.S. Pat. No. 6,670,355 discloses the useof rapamycin in treating cardiovascular, cerebral vascular, orperipheral vascular disease; U.S. Pat. No. 5,561,138 discloses the useof rapamycin in treating immune related anemia; U.S. Pat. No. 5,288,711discloses a method of preventing or treating hyperproliferative vasculardisease including intimal smooth muscle cell hyperplasia, restenosis,and vascular occlusion with rapamycin; and U.S. Pat. No. 5,321,009discloses the use of rapamycin in treating insulin dependent diabetesmellitus.

H. Pharmaceutical Preparations

Certain methods and compositions set forth herein are directed toadministration of an effective amount of a composition comprising therapamycin compositions of the present invention.

1. Compositions

A “pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, surfactants, antioxidants, preservatives(e.g., antibacterial agents, antifungal agents), isotonic agents,absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (Remington's, 1990). Except insofar as any conventionalcarrier is incompatible with the active ingredient, its use in thetherapeutic or pharmaceutical compositions is contemplated. Thecompositions used in the present invention may comprise different typesof carriers depending on whether it is to be administered in solid,liquid or aerosol form, and whether it needs to be sterile for suchroutes of administration as injection.

The use of such media and agents for pharmaceutically active substancesis well known in the art. Except insofar as any conventional media oragent is incompatible with the active ingredient, its use in thetherapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions, and theseare discussed in greater detail below. For human administration,preparations should meet sterility, pyrogenicity, general safety andpurity standards as required by FDA Office of Biologics standards.

The formulation of the composition may vary depending upon the route ofadministration. For parenteral administration in an aqueous solution,for example, the solution should be suitably buffered if necessary andthe liquid diluent first rendered isotonic with sufficient saline orglucose. In this connection, sterile aqueous media that can be employedwill be known to those of skill in the art in light of the presentdisclosure.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other pharmaceuticallyacceptable forms include, e.g., tablets or other solids for oraladministration; liposomal and nanoparticle formulations; enteric coatingformulations; time release capsules; formulations for administration viaan implantable drug delivery device, and any other form. One may alsouse nasal solutions or sprays, aerosols or inhalants in the presentinvention.

The capsules may be, for example, hard shell capsules or soft-shellcapsules. The capsules may optionally include one or more additionalcomponents that provide for sustained release.

In certain embodiments, pharmaceutical composition includes at leastabout 0.1% by weight of the active compound. In other embodiments, thepharmaceutical composition includes about 2% to about 75% of the weightof the composition, or between about 25% to about 60% by weight of thecomposition, for example, and any range derivable therein.

The compositions may comprise various antioxidants to retard oxidationof one or more components. Additionally, the prevention of the action ofmicroorganisms can be accomplished by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof. The composition shouldbe stable under the conditions of manufacture and storage, and preservedagainst the contaminating action of microorganisms, such as bacteria andfungi.

In certain embodiments, an oral composition may comprise one or morebinders, excipients, disintegration agents, lubricants, flavoringagents, and combinations thereof. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type,carriers such as a liquid carrier. Various other materials may bepresent as coatings or to otherwise modify the physical form of thedosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

In particular embodiments, prolonged absorption can be brought about bythe use in the compositions of agents delaying absorption, such as, forexample, aluminum monostearate, gelatin, or combinations thereof.

2. Routes of Administration

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective.

The composition can be administered to the subject using any methodknown to those of ordinary skill in the art. For example, apharmaceutically effective amount of the composition may be administeredintravenously, intracerebrally, intracranially, intraventricularly,intrathecally, into the cortex, thalamus, hypothalamus, hippocampus,basal ganglia, substantia nigra or the region of the substantia nigra,cerebellum, intradermally, intraarterially, intraperitoneally,intralesionally, intratracheally, intranasally, topically,intramuscularly, intraperitoneally, anally, subcutaneously, orally,topically, locally, inhalation (e.g., aerosol inhalation), injection,infusion, continuous infusion, localized perfusion bathing target cellsdirectly, via a catheter, via a lavage, in creams, in lipid compositions(e.g., liposomes), or by other method or any combination of the forgoingas would be known to one of ordinary skill in the art (Remington's,1990).

In particular embodiments, the composition is administered to a subjectusing a drug delivery device. Any drug delivery device is contemplatedfor use in delivering an effective amount of the inhibitor of mTORC 1.

3. Dosage

A pharmaceutically effective amount of an inhibitor of mTORC1 isdetermined based on the intended goal. The quantity to be administered,both according to number of treatments and dose, depends on the subjectto be treated, the state of the subject, the protection desired, and theroute of administration. Precise amounts of the therapeutic agent alsodepend on the judgment of the practitioner and are peculiar to eachindividual.

The amount of rapamycin or rapamycin analog or derivative to beadministered will depend upon the disease to be treated, the length ofduration desired and the bioavailability profile of the implant, and thesite of administration. Generally, the effective amount will be withinthe discretion and wisdom of the patient's physician. Guidelines foradministration include dose ranges of from about 0.01 mg to about 500 mgof rapamycin or rapamycin analog.

For example, a dose of the inhibitor of mTORC1 may be about 0.0001milligrams to about 1.0 milligrams, or about 0.001 milligrams to about0.1 milligrams, or about 0.1 milligrams to about 1.0 milligrams, or evenabout 10 milligrams per dose or so. Multiple doses can also beadministered. In some embodiments, a dose is at least about 0.0001milligrams. In further embodiments, a dose is at least about 0.001milligrams. In still further embodiments, a dose is at least 0.01milligrams. In still further embodiments, a dose is at least about 0.1milligrams. In more particular embodiments, a dose may be at least 1.0milligrams. In even more particular embodiments, a dose may be at least10 milligrams. In further embodiments, a dose is at least 100 milligramsor higher.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

The dose can be repeated as needed as determined by those of ordinaryskill in the art. Thus, in some embodiments of the methods set forthherein, a single dose is contemplated. In other embodiments, two or moredoses are contemplated. In some embodiments, the two or more doses arethe same dosage. In some embodiments, the two or more doses aredifferent dosages. Where more than one dose is administered to asubject, the time interval between doses can be any time interval asdetermined by those of ordinary skill in the art. For example, the timeinterval between doses may be about 1 hour to about 2 hours, about 2hours to about 6 hours, about 6 hours to about 10 hours, about 10 hoursto about 24 hours, about 1 day to about 2 days, about 1 week to about 2weeks, or longer, or any time interval derivable within any of theserecited ranges. In specific embodiments, the composition may beadministered daily, weekly, monthly, annually, or any range therein.

Doses for encapsulated rapamycin (eRapa) and for encapsulated rapamycinnanoparticles maybe different. According to certain embodiments, dosesare contemplated in a range of more than 50 micrograms and up to (oreven exceeding) 200 micrograms per kilogram for daily administration, orthe equivalent for other frequencies of administration. Although dosingmay vary based on particular needs and preferred treatment protocolsaccording to physician preference, maximum tolerable daily bioavailabledosings (trough levels) for a 28-day duration are about 200 microgramsof rapamycin (or equivalent) per subject kilogram, for both human andcanine subjects, although those of ordinary skill would understand thatgreater dose amount ranges would be tolerable and suitable whenadministered less often than once per day, and lesser ranges would betolerable when administered more often than once per day.

In certain embodiments, it may be desirable to provide a continuoussupply of a pharmaceutical composition to the patient. This could beaccomplished by catheterization, followed by continuous administrationof the therapeutic agent. The administration could be intra-operative orpost-operative.

4. Secondary and Combination Treatments

Certain embodiments provide for the administration or application of oneor more secondary or additional forms of therapies. The type of therapyis dependent upon the type of disease that is being treated orprevented. The secondary form of therapy may be administration of one ormore secondary pharmacological agents that can be applied in thetreatment or prevention of intestinal polyps or cancer or a disease,disorder, or condition associated with intestinal polyps and cancer in apatient who has been identified as being at risk for developingintestinal polyps or intestinal cancer.

If the secondary or additional therapy is a pharmacological agent, itmay be administered prior to, concurrently, or following administrationof the inhibitor of mTORC1.

The interval between administration of the inhibitor of mTORC1 and thesecondary or additional therapy may be any interval as determined bythose of ordinary skill in the art. For example, the inhibitor of mTORC1and the secondary or additional therapy may be administeredsimultaneously, or the interval between treatments may be be minutes toweeks. In embodiments where the agents are separately administered, onewould generally ensure that a significant period of time did not expirebetween the time of each delivery, such that each therapeutic agentwould still be able to exert an advantageously combined effect on thesubject. For example, the interval between therapeutic agents may beabout 12 h to about 24 h of each other or within about 6 hours to about12 h of each other. In some situations, it may be desirable to extendthe time period for treatment significantly, however, where several days(2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapsebetween the respective administrations. In some embodiments, the timingof administration of a secondary therapeutic agent is determined basedon the response of the subject to the inhibitor of mTORC1.

I. Kits

Kits are also contemplated as being used in certain aspects of thepresent invention. For instance, a rapamycin composition of the presentinvention can be included in a kit. A kit can include a container.Containers can include a bottle, a metal tube, a laminate tube, aplastic tube, a dispenser, a pressurized container, a barrier container,a package, a compartment, or other types of containers such as injectionor blow-molded plastic containers into which the hydrogels are retained.The kit can include indicia on its surface. The indicia, for example,can be a word, a phrase, an abbreviation, a picture, or a symbol.

Further, the rapamycin compositions of the present invention may also besterile, and the kits containing such compositions can be used topreserve the sterility. The compositions may be sterilized via anaseptic manufacturing process or sterilized after packaging by methodsknown in the art.

EXAMPLES

The following examples are included to demonstrate certain non-limitingaspects of the invention. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent techniques discovered by the inventors to function well in thepractice of the invention. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1

Encapsulated rapamycin, sometimes referred to as eRapa, increases lifespan in a mouse model of colon cancer, referred to as Apc^(Min/+). Thismouse model carries a germ line mutation in one copy of the mouse tumorsuppressor gene encoding adenomatous polyposis coli (Apc). Min inApc^(Min/+) refers to a condition called multiple intestinal neoplasms,which in this mouse model develop very early in life, resulting in ashort life span of about 180 days. The cause of death of Apc^(Min/−)mice is usually severe anemia due to bleeding from the multipleneoplastic polyps in the intestine. Apc^(Min/+) mice model in part aninherited condition in humans called familial adenomatous polyposis(FAP).

Familial adenomatous polyposis (FAP) is an autosomal dominant diseasecaused by mutation of the Adenomatous Polyposis Coli (APC) gene, locatedon chromosome 5 (Kinzler, 1991). This germline defect accelerates theinitiation of adenoma-carcinoma, resulting in the development ofnumerous adenomatous colorectal polyps at a young age. Polyposisinevitably progresses to colorectal cancer if left untreated. Given thepredictable development of colorectal cancer in patients with FAP, thesafest preventative strategy is surgical resection of the colon whenpolyposis develops. The two main prophylactic surgeries are colectomywith ileorectal anastamosis (IRA) and proctocolectomy with ilealpouch-anal anastamosis (IPAA) (Vasen, 2008). Genetic screening andendoscopy in concert with prophylactic surgery significantly improvedthe overall survival of FAP patients. However, less well appreciated bymedical providers is the second leading cause of death in FAP, duodenaladenocarcinoma. Nearly 90% of patients with FAP develop duodenal polyps,the precursor lesions of duodenal adenocarcinoma (Wallace, 1998) and4.5% will develop duodenal adenocarcinoma in their lifetime (Bulow,2004). In contrast to the colon, prophylactic surgical resection of theampulla and/or duodenum is accompanied by significant morbidity.Duodenal surgery is currently indicated for patients with severeduodenal polyposis or duodenal carcinoma. This patient population has astrong need for adjuvant therapies to surgery to prevent or reduce thepolyp formation and carcinogenesis in the gastro-intestinal track.

Since FAP patients develop polyps that eventually progress to coloncancer, and since Apc^(Min/+) mice develop similar neoplasms, this mousemodel (and other containing similar mutations in Apc) are widely used byintestinal cancer researchers.

Showing that eRapa treatment beginning early in life in Apc^(Min/+) miceprevents polyps from developing and progressing to the bleeding stagethereby resulting in a life span equal to and perhaps greater than wildtype, normal mice (See FIG. 1-A-2 for comparison to life span of normal,wild type) strongly suggests a similar approach in FAP (and other typesof GI cancers) in human patients will be of great benefit.

Apc^(Min/+) mice were fed Eudragit control chow (0 ppm rapamycin), amedium dose of 14 ppm encapsulated rapamycin (2.24 mg/kg/day), or a highdose of 42 ppm encapsulated rapamycin (6.72 mg/kg/day) chow beginning at6 weeks of age (FIG. 1(A), arrow). All mice consuming 0 ppm chow died by181 days of age, while the rapamycin-treated mice survived to between570 and 685 days (median 668 days) for the mice dosed at 14 ppm and tobetween 559 days to 1,093 days (median 937 days). This extension of lifespan was statistically significant (Logrank Test; p<0.0025) for eachdose. FIG. 1(B) In FIG. 1(B), life span of eRapa-treated Apc^(Min/+)mice was compared to wild type C57BL6 mice or mice treated with RAD001(everolimus), as reported by Fujishita T, Aoki K, Lane H A, Aoki M,Taketo M M in “Inhibition of the mTORC1 pathway suppresses intestinalpolyp formation and reduces mortality in Apc^(Δ716) mice” (published inProc. Natl. Acad. Sci. USA, 2008 Sep. 9; 105(36):13544-9). Thisexperiment reveals that eRapa is more effective than the RAD001treatment because the lower dose of eRapa (2.24 mg/kg) results in alonger life span than the highest dose of RAD001 (10 mg/kg). Inaddition, 60% of the Apc^(Min/+) mice receiving 42 ppm eRapa diets livedbeyond mice treated with the highest dose of RAD001 and wild type,normal mice.

Intestinal polyp counts of Apc^(Min/+) mice at necropsy show areduction, especially the mice treated with high dose. The first mouseto die in the high dose treatment group showed no visible signs ofintestinal neoplasms. The second mouse that died, had three polyps. Thisis evidence of prevention of neoplastic disease in a highly prone mousemodel.

Encapsulated rapamycin also improves the health of the treated mice. Thehealth of the mice was tested by monitoring their activity. Older orsick mice move less than younger, healthy mice. The Nathan ShockHealthspan and Functional Assessment Core of the Barshop Institute forLongevity and Aging Studies documented the activity of therapamycin-treated and control mice. The data shown in FIG. 2(B) revealthe decline in movement by the 0 ppm fed group (labeled control in thegraphs), which has been prevented by both the medium (2.24 mg/kg/day)and high (6.72 mg/kg/day) doses of rapamycin. Both the mid and high doseare equally effective in maintaining this aspect of health. The datashown in FIG. 2(B) also show a difference between movement between lightand dark phases of the day cycle for the medium and high doses ofrapamycin, the difference being absent in the control 0 ppm dose mice.These data indicate the maintenance of a diurnal rhythm and activitylevels similar to wild-type C57BL/6 mice, suggesting better healthversus Apc^(Min/+) mice on control chow.

FIG. 2(A) demonstrates that polyp count at the time of death was lowerin Apc^(Min/+) mice that were treated with eRapa. The first mouse thatdied after treatment with 42 ppm eRapa had no polyps, and the second onehad only 3 polyps. FIG. 2(C) demonstrates that encapsulated Rapamaintains normal hematocrits in Apc^(Min/+) mice. The hematocrit ineRapa-treated mice (in the high dose group) was normal as compared towild type C57BL/6 mice (44%) even at 550 days, a time when about 5% ofwild type C57BL/6 mice were reported to die from natural causes. It isclear that the high dose eRapa is more effective in maintaining normalhematocrits, which is indicative of the repression of mTORC1 (shown inFIG. 3) and inhibition of polyp development and growth leading toextended longevity in this tumor-prone model.

FIG. 3(A&B) shows a dose-dependent depression of the phosphorylation ofrpS6 by chronic eRapa treatment. rpS6 was recently shown to have a vitalrole in ribosome biogenesis needed for protein synthesis, developmentand growth of intestinal neoplasms. Chauvin C, Koka V, Nouschi A,Mieulet V, Hoareau-Aveilla C, Dreazen A, et al, Oncogene, 2013. Both midand high doses are equally effective in repressing this part of mTORC1downstream signaling.

Also shown are blood levels of rapamycin by the 2.24 mg/kg and 6.72mg/kg eRapa doses (FIG. 3(C). These blood concentrations are higher thanthe therapeutic range used for organ transplant recipients. Trepanier D,Gallant H, Legatt D, Yatscoff R. Clin Biochem 1998, 31:345-351. A doseresponse was observed in proximal and distal small intestine tissuelevels of rapamycin, the increase in the distal intestine compared withthe proximal, which is consistent with the pH gradient approachingneutrality thereby resulting in an increase drug release by Eudragitdelivery. This implies that eRapa may be an effective and convenientmethod to deliver rapamycin to both the small intestine and blood,indicating that eRapa may have both local and systemic effects.

These data of increased lifespan, increased activity levels, increasedhematocrit, dose-dependent depression of the phosphorylation of rpS6,decreased polyp production, and other health indicators is not dueexclusively to the dose of rapamycin delivered in the chow. The low doseof 2.24 mg/kg/day is lower than other reported doses of rapamycin suchas 3 mg/kg/day and 10 mg/kg/day by oral gavage (Fujishita, et al., ProcNatl Acad Sci USA. 105(36):13544-9, 2008) and 40 mg/kg food pellet(Koehl, et al., Oncogene, 29:1553-60, 2010). However, and surprisingly,the rapamycin encapsulated in the Eudragit provided a superiortherapeutic benefit than rapamycin delivered alone.

The studies described above demonstrate that encapsulated rapamycin inthe disclosed formulation, which is enterically delivered, prevents,delays the development of, or slows the growth and progression ofintestinal polyps (and subsequent cancer) in this mouse model.

Example 2

Development of methods to produce rapamycin nanoparticles. Rapid solventexchange was used to examine the formation of rapamycin nanoparticles.Three water-miscible solvents and three water-soluble surfactants wereselected to study their respective effects on the formation andmorphology of rapamycin nanoparticles. The water-miscible solvents wereisopropyl alcohol (Solvent 1), acetone (Solvent 2), and methanol(Solvent 3). The water-soluble surfactants were Pluronic F-68(Dispersant 1, a non-ionic PEO-PPO-PEO block copolymer), Pluronic F-127(Dispersant 2, a non-ionic PEO-PPO-PEO block copolymer), and sodiumcholate (Dispersant 3, an anionic surfactant). Rapamycin was dissolvedin each of the water-miscible solvents at a concentration of 0.25% w/v.The water-soluble surfactants were dissolved in deionized water atconcentrations of 0.5% w/v, 0.5% w/v, and 1.0% w/v, respectively, foreach of the dispersants. Each experimental combination (e.g. NP-1 toNP-9 in following table) consisted of 5 mL of rapamycin solution and 25mL of surfactant solution, resulting in a dilution factor of 1:5solvent:water. 25 mL of surfactant solution was transferred to a 50 mLbeaker and stirred with the aid of magnetic mircostirbar. Rapamycinsolution was rapidly injected at 500 uL increments with the aid of amicropipette with the pipette tip placed below the surface of therapidly stirred surfactant solution. The visual appearance of theresulting nanoparticles and their colloidal stability after 24-hourswere qualitatively assessed. The following table summarizes thequalities of the rapamycin nanoparticle dispersions. Qualitatively,rapamycin nanoparticle dispersions having a colorless to blue,opalescent appearance will have particle sizes on the order of less thanabout 300 nm as evidenced by their interaction with the ultravioletwavelengths of visible light. Whereas, dispersions having a more whiteappearance will have particle sizes larger than about 300 nm due totheir interaction with the broader spectrum of visible light. Rapamycinnanoparticle formulations NP-7 and NP-9 were selected as methods ofnanoparticle preparation.

Dispersant 1 Dispersant 2 Dispersant 3 Solvent 1 NP-1: White, settled,NP-2: Blue, NP-3: Clear, resdispersible opalescent, settled, aggregated,redispersible redispersible Solvent 2 NP-4: Blue, NP-5: White, settled,NP-6: Blue, opalescent, some redispersible opalescent, settling settled,redispersible Solvent 3 NP-7: Blue, NP-8: Blue to white, NP-9: Blue,opalescent, stable settled, redispersible opalescent, stable

Example 3

Preparation of a high concentration rapamycin nanoparticle dispersion.The water-miscible solvent and water-soluble dispersant of NP-9 fromExample 1 was used to prepare rapamycin nanoparticles. 656 mg ofrapamycin were dissolved in 6.56 mL of Solvent 3 to yield a 1.0% w/vsolution. This volume of rapamycin solution was injected into 26.25 mLof 1.0% w/v Dispersant 1 in deionized water. The resulting rapamycinnanoparticle dispersion had a final rapamycin content of 2.4% w/w. Theparticle size of the dispersion was determined by dynamic lightscattering to be 230 nm±30 nm with a single peak.

Example 4

Preparation of a water-soluble enteric coating. 3.5 g of EUDRAGIT® S-100were added to 70 mL of deionized water with light stirring, resulting ina white dispersion. 1.4 g of sodium hydroxide were added to thedispersion with continued stirring. The resulting dispersion graduallyturned clear and colorless indicating an aqueous solution of S-100. Theestimated concentration of sodium hydroxide was 0.5N.

Example 5

Preparation of a feedstock containing rapamycin nanoparticles and awater-soluble enteric coating. Rapamycin nanoparticles were prepared asdescribed in Example 2 and then slowly added to an aqueous solution ofEUDRAGIT® S-100 prepared as in Example 3. The ratio of rapamycin toS-100 was 1:9, or 10% wt. rapamycin payload. The resulting dispersionwas allowed to stir for several minutes to observe stability. After onehour, the dispersion had transformed to a clear yellow, indicatingdestruction of the rapamycin nanoparticles and a change in therapamycin. Addition of a small amount of acetic acid to reduce thesolution pH to below neutral resulted in a clear, colorless solution.

Example 6

Preparation of water-soluble enteric coating with a water-miscibleco-solvent. 3.5 g of EUDRAGIT® S-100 were added to 30/70 v/vmethanol/deionized water, resulting in a white dispersion. Thedispersion was stirred continuously until a clear solution was formed.

Example 7

Preparation of a feedstock containing rapamycin nanoparticles and awater-soluble enteric coating. Rapamycin nanoparticles were prepared asdescribed in Example 2 and then slowly added to an aqueous solution ofEUDRAGIT® S-100 prepared as in Example 5. The ratio of rapamycin toS-100 was 1:9, or 10% wt. rapamycin payload. The white dispersion wasallowed to stir for several minutes after which the dispersion wastransformed into a clear solution indicating the rapamycin nanoparticleshad been destroyed.

Example 8

Preparation of a partially-neutralized, water-soluble enteric coatingwith a water-miscible co-solvent. 3.5 g of EUDRAGIT® S-100 were added to10/90 v/v methanol/deionized water, resulting in a white dispersion. Thedispersion was titrated to clarity with 2.000 mL of 4.8M sodiumhydroxide. The estimated neutralization of the S-100 was 78%.

Example 9

Preparation of a feedstock containing rapamycin nanoparticles and awater-soluble enteric coating. Rapamycin nanoparticles were prepared asdescribed in Example 2 then slowly added to an aqueous solution ofEUDRAGIT® S-100 as prepared in Example 7. The ratio of rapamycin toS-100 was 1:9, or 10% wt. rapamycin payload. The resulting whitedispersion remained stable for several hours as indicated by no changein color or change in optical clarity. The final pH was 7.5. Theparticle size of the final dispersion was determined by dynamic lightscattering to be 756 nm±52 nm with a single peak and indicating possibleclustering of the rapamycin nanoparticles in the resulting feedstock.

Example 10

Preparation of a feedstock containing rapamycin nanoparticles and awater-soluble enteric coating. The rapamycin solution used in Example 2was injected with stirring into the aqueous solution of EUDRAGIT® S-100prepared in Example 7. The ratio of rapamycin to S-100 was 1:9, or 10%wt. rapamycin payload. A blue, opalescent colloid was formed and itremained stable for several hours as indicated by no change in color orchange in optical clarity. The final pH was 7.5. The particle size ofthe final dispersion was determined by dynamic light scattering to be305 nm±60 nm with a single peak.

Example 11

Spray drying of feedstock containing rapamycin nanoparticles and awater-soluble enteric coating. The feedstocks prepared in Examples 8 and9 were spray dried and analyzed for rapamycin content. Particlesprepared from Example 8 had a rapamycin content of 9.5% wt. (87%rapamycin yield). Particles prepared from Example 9 had a rapamycincontent of 7.9% wt. (80% rapamycin yield).

Example 12

Storage stability of enteric-coated encapsulated rapamycinnanoparticles. Microparticles prepared by spray drying in Example 10were stored under controlled conditions at room temperature and 50%relative humidity. Samples were analyzed weekly for rapamycin content.All samples maintained at least 95% of their original rapamycin contentat all time points for at least three weeks.

Example 13

Preparation of nanoparticles in Eudragit S-100. Referring to FIG. 7, arapamycin solution was prepared by combining rapamycin with methanol ina 10% w/v ratio as 3.03 g rapamycin and 30.25 ml methanol. A 1% w/wsodium cholate solution was prepared by combining 1.2 g sodium cholatewith 120 ml deionized water. Nanoparticle formation was achieved bytransferring the rapamycin solution with a 60 ml plastic syringeequipped with a 20 ga needle, injecting the rapamycin solution below thesurface of the sodium cholate solution in a 250 ml beaker. Mixing wasaccomplished with a paddle mixer operating at 300 rpm yielding arapamycin nanoparticle suspension. A 10% w/w Eudragit S-100 solution wasprepared by combining 20 g Eudragit S-100 in a 9.7% w/v mixture with 180ml deionized water, 25.72 ml methanol in a 12.5% v/v mixture, and 1.8 gsodium cholate in a 0.875% w/v mixture. This 10% w/w Eudragit S-100solution was titrated with 4M sodium hydroxide to achieve a pH ofbetween about 7.5 and about 7.6. Encapsulated rapamycin particles werethen fabricated by combining the Eudragit S-100 solution with therapamycin nanoparticle suspension. The Eudragit 5-100 solution and therapamycin nanoparticle suspension were combined in a 500 ml bottle,adding 2.13 g of glycerol and mixing with a magnetic stir bar. Thecombined Eudragit S-100 solution and rapamycin nanoparticle suspensionwere then spray dried and collected. The spray drying parametersincluded a 0.4 mm nozzle, nozzle air pressure of 3 bar, input airtemperature of 110° C., a sample pump rate of 5 ml/min and an air speedof 0.30 m3/min.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method for preventing intestinal polyps or intestinal cancer in apatient comprising administering an effective amount of a compositioncomprising rapamycin or an analog thereof to a patient who has beenidentified as being at risk for developing intestinal polyps orintestinal cancer.
 2. The method of claim 1, wherein the rapamycin oranalog thereof is encased in a coating comprising cellulose acetatesuccinate, hydroxy propyl methyl cellulose phthalate co-polymer, or apolymethacrylate-based copolymer selected from the group consisting ofmethyl acrylate-methacrylic acid copolymer, and a methylmethacrylate-methacrylic acid copolymer.
 3. The method of claim 2,wherein the coating comprises Poly(methacylic acid-co-ethyl acrylate) ina 1:1 ratio, Poly(methacrylic acid-co-ethyl acrylate) in a 1:1 ratio,Poly(methacylic acid-co-methyl methacrylate) in a 1:1 ratio,Poly(methacylic acid-co-methyl methacrylate) in a 1:2 ratio, Poly(methylacrylate-co-methyl methacrylate-co-methacrylic acid) in a 7:3:1 ratio,Poly(ethyl acrylate-co-methyl methacrylate-co-trimethylammonioethylmethacrylate chloride) in a 1:2:0.2 ratio, Poly(ethyl acrylate-co-methylmethacrylate-co-trimethylammonioethyl methacrylate chloride) in a1:2:0.1 ratio, or Poly(butyl methacylate-co-(2-dimethylaminoethyl)methacrylate-co-methyl methacrylate) in a 1:2:1 ratio, anaturally-derived polymer, or a synthetic polymer, or any combinationthereof.
 4. The method of claim 3, wherein the naturally-derived polymeris selected from the group consisting of alginates and their variousderivatives, chitosans and their various derivatives, carrageenans andtheir various analogues, celluloses, gums, gelatins, pectins, gellans,polyethyleneglycols (PEGs) and polyethyleneoxides (PEOs), acrylic acidhomo- and copolymers with acrylates and methacrylates, homopolymers ofacrylates and methacrylates, polyvinyl alcohol PVOH), and polyvinylpyrrolidone (PVP).
 5. (canceled)
 6. The method of claim 1, wherein thepatient has been diagnosed with an inflammatory bowel disease.
 7. Themethod of claim 1, wherein the patient has been diagnosed with anintestinal polyp or an adenoma.
 8. The method of claim 1, wherein thepatient has been diagnosed as having a mutation that is known to causeincreased WNT signaling.
 9. The method of claim 1, wherein the patienthas been diagnosed as having Familial Adenomatous Polyposis (FAP). 10.The method of claim 1, wherein the patient has a family history ofintestinal polyps or intestinal cancer.
 11. The method of claim 1,wherein the patient is between the ages of 1 to 18 years, 18 years to 50years, or over the age of 50 years.
 12. The method of claim 1, whereinthe composition comprises rapamycin or an analog thereof at aconcentration of 0.001 mg to 30 mg total per dose.
 13. The method ofclaim 1, wherein the composition comprising rapamycin or an analog ofrapamycin comprises 0.001% to 60% by weight of rapamycin or an analog ofrapamycin.
 14. The method of claim 1, wherein the average blood level ofrapamycin in the subject is greater than 0.5 ng per mL whole blood afteradministration of the composition.
 15. The method of claim 1, whereinthe composition is administered orally, enterically, colonically,anally, intravenously, or dermally with a patch. 16-18. (canceled) 19.The method of claim 1, wherein the rapamycin or analog of rapamycin isadministered in two or more doses; and wherein the interval of timebetween administration of doses comprising rapamycin or an analog ofrapamycin is 1 to 3 days. 20-22. (canceled)
 23. The method of claim 1,wherein the subject is further administered a composition comprising asecond active agent, wherein the second active agent is metformin,celocoxib, eflornithine, sulindac, ursodeoxycholic acid, ananti-inflammatory agent, an anti-autoimmune agent, or a cytotoxic orcytostatic anti-cancer agent.
 24. (canceled)
 25. The method of claim 23,wherein the composition comprising rapamycin or an analog of rapamycinis administered at the same time as the composition comprising thesecond active agent.
 26. The method of claim 23, wherein the compositioncomprising rapamycin or an analog of rapamycin is administered before orafter the composition comprising the second active agent isadministered; and wherein the interval of time between administration ofcomposition comprising rapamycin or an analog of rapamycin and thecomposition comprising the second active agent is 1 to 30 days. 27-28.(canceled)
 29. The method of claim 1, wherein the composition comprisingrapamycin or an analog of rapamycin prevents the development of newadenomas or polyps, decreases the number or severity of the adenomatouspolyps, induces a reduction in size or number of existing adenomas orpolyps, prevents the conversion of adenomas or polyps intoadenocarcinomas and cancer tissue, or prevents the adenomas or polypsfrom converting into malignant cancer that spread into other bodilytissues, organs and blood systems in a patient that has been diagnosedas having intestinal adenomas, intestinal polyps or Familial AdenomatousPolyposis (FAP).
 30. The method of claim 1, wherein the compositioncomprising rapamycin or an analog of rapamycin is comprised in a food orfood additive.